Abstract
Labeling with deuterium oxide (D(2)O) has emerged as one of the preferred approaches for measuring the synthesis of individual proteins in vivo. In these experiments, the synthesis rates of proteins are determined by modeling mass shifts in peptides during the labeling period. This modeling depends on a theoretical maximum enrichment determined by the number of labeling sites (N(EH)) of each amino acid in the peptide sequence. Currently, N(EH) is determined from one set of published values. However, it has been demonstrated that N(EH) can differ between species and potentially tissues. The goal of this work was to determine the number of N(EH) for each amino acid within a given experiment to capture the conditions unique to that experiment. We used four methods to compute the N(EH) values. To test these approaches, we used two publicly available data sets. In a de novo approach, we compute N(EH) values and the label enrichment from the abundances of three mass isotopomers. The other three methods use the complete isotope profiles and body water enrichment in deuterium as an input parameter. They determine the N(EH) values by (1) minimizing the residual sum of squares, (2) from the mole percent excess of labeling, and (3) the time course profile of the depletion of the relative isotope abundance of monoisotope. In the test samples, the method using residual sum of squares performed the best. The methods are implemented in a tool for determining the N(EH) for each amino acid within a given experiment to use in the determination of protein synthesis rates using D(2)O.